"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies

European Cross-Sectional Survey of Current Care Practices for Duchenne Muscular Dystrophy Reveals Regional and Age-Dependent Differences

BACKGROUND: Publication of comprehensive clinical care guidelines for Duchenne muscular dystrophy (DMD) in 2010 was a milestone for DMD patient management. Our CARE-NMD survey investigates the neuromuscular, medical, and psychosocial care of DMD patients in Europe, and compares it to the guidelines. METHODS: A cross-sectional survey of 1677 patients contacted via the TREAT-NMD patient registries was conducted using self-report questionnaires in seven European countries. RESULTS: Survey respondents were 861 children and 201 adults. Data describe a European DMD population with mean age of 13.0 years (range 0.8-46.2) of whom 53% had lost ambulation (at 10.3 years of age, median). Corticosteroid medication raised the median age for ambulatory loss from 10.1 years in patients never medicated to 11.4 years in patients who received steroids (p /= nine years received no corticosteroid medication, 24% of all patients received no regular physiotherapy, echocardiograms were not performed regularly in 22% of patients, pulmonary function was not regularly assessed in 71% of non-ambulatory patients. Patients with regular follow-up by neuromuscular specialists were more likely to receive care according to guidelines, were better satisfied, and experienced shorter unplanned hospitalization periods

Creation of a novel algorithm to identify patients with Becker and Duchenne muscular dystrophy within an administrative database and application of the algorithm to assess cardiovascular morbidity

BACKGROUND: Outcome analyses in large administrative databases are ideal for rare diseases such as Becker and Duchenne muscular dystrophy. Unfortunately, Becker and Duchenne do not yet have specific International Classification of Disease-9/-10 codes. We hypothesised that an algorithm could accurately identify these patients within administrative data and improve assessment of cardiovascular morbidity. METHODS: Hospital discharges (n=13,189) for patients with muscular dystrophy classified by International Classification of Disease-9 code: 359.1 were identified from the Pediatric Health Information System database. An identification algorithm was created and then validated at three institutions. Multi-variable generalised linear mixed-effects models were used to estimate the associations of length of stay, hospitalisation cost, and 14-day readmission with age, encounter severity, and respiratory disease accounting for clustering within the hospital. RESULTS: The identification algorithm improved identification of patients with Becker and Duchenne from 55% (code 359.1 alone) to 77%. On bi-variate analysis, left ventricular dysfunction and arrhythmia were associated with increased cost of hospitalisation, length of stay, and mortality (p<0.001). After adjustment, Becker and Duchenne patients with left ventricular dysfunction and arrhythmia had increased length of stay with rate ratio 1.4 and 1.2 (p<0.001 and p=0.004) and increased cost of hospitalization with rate ratio 1.4 and 1.4 (both p<0.001). CONCLUSIONS: Our algorithm accurately identifies patients with Becker and Duchenne and can be used for future analysis of administrative data. Our analysis demonstrates the significant effects of cardiovascular disease on length of stay and hospitalisation cost in patients with Becker and Duchenne. Better recognition of the contribution of cardiovascular disease during hospitalisation with earlier more intensive evaluation and therapy may help improve outcomes in this patient population

Poloxomer 188 Has a Deleterious Effect on Dystrophic Skeletal Muscle Function

Duchenne muscular dystrophy (DMD) is an X-linked, fatal muscle wasting disease for which there is currently no cure and limited palliative treatments. Poloxomer 188 (P188) is a tri-block copolymer that has been proposed as a potential treatment for cardiomyopathy in DMD patients. Despite the reported beneficial effects of P188 on dystrophic cardiac muscle function, the effects of P188 on dystrophic skeletal muscle function are relatively unknown. Mdx mice were injected intraperitoneally with 460 mg/kg or 30 mg/kg P188 dissolved in saline, or saline alone (control). The effect of single-dose and 2-week daily treatment was assessed using a muscle function test on the Tibialis Anterior (TA) muscle in situ in anaesthetised mice. The test comprises a warm up, measurement of the force-frequency relationship and a series of eccentric contractions with a 10% stretch that have previously been shown to cause a drop in maximum force in mdx mice. After 2 weeks of P188 treatment at either 30 or 460 mg/kg/day the drop in maximum force produced following eccentric contractions was significantly greater than that seen in saline treated control mice (P = 0.0001). Two week P188 treatment at either dose did not significantly change the force-frequency relationship or maximum isometric specific force produced by the TA muscle. In conclusion P188 treatment increases susceptibility to contraction-induced injury following eccentric contractions in dystrophic skeletal muscle and hence its suitability as a potential therapeutic for DMD should be reconsidered

Membrane Sealant Poloxamer P188 Protects Against Isoproterenol Induced Cardiomyopathy in Dystrophin Deficient Mice

<p>Abstract</p> <p>Background</p> <p>Cardiomyopathy in Duchenne muscular dystrophy (DMD) is an increasing cause of death in patients. The absence of dystrophin leads to loss of membrane integrity, cell death and fibrosis in cardiac muscle. Treatment of cardiomyocyte membrane instability could help prevent cardiomyopathy.</p> <p>Methods</p> <p>Three month old female mdx mice were exposed to the β₁receptor agonist isoproterenol subcutaneously and treated with the non-ionic tri-block copolymer Poloxamer P188 (P188) (460 mg/kg/dose i.p. daily). Cardiac function was assessed using high frequency echocardiography. Tissue was evaluated with Evans Blue Dye (EBD) and picrosirius red staining.</p> <p>Results</p> <p>BL10 control mice tolerated 30 mg/kg/day of isoproterenol for 4 weeks while death occurred in mdx mice at 30, 15, 10, 5 and 1 mg/kg/day within 24 hours. Mdx mice tolerated a low dose of 0.5 mg/kg/day. Isoproterenol exposed mdx mice showed significantly increased heart rates (p < 0.02) and cardiac fibrosis (p < 0.01) over 4 weeks compared to unexposed controls. P188 treatment of mdx mice significantly increased heart rate (median 593 vs. 667 bpm; p < 0.001) after 2 weeks and prevented a decrease in cardiac function in isoproterenol exposed mice (Shortening Fraction = 46 ± 6% vs. 35 ± 6%; p = 0.007) after 4 weeks. P188 treated mdx mice did not show significant differences in cardiac fibrosis, but demonstrated significantly increased EBD positive fibers.</p> <p>Conclusions</p> <p>This model suggests that chronic intermittent intraperitoneal P188 treatment can prevent isoproterenol induced cardiomyopathy in dystrophin deficient mdx mice.</p

Glucocorticoid-Treated Mice Are an Inappropriate Positive Control for Long-Term Preclinical Studies in the mdx Mouse

Dmd(mdx) (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials

Skeletal, cardiac, and respiratory muscle function and histopathology in the P448Lneo- mouse model of FKRP-deficient muscular dystrophy.

BACKGROUND: Fukutin-related protein (FKRP) mutations are the most common cause of dystroglycanopathies known to cause both limb girdle and congenital muscular dystrophy. The P448Lneo- mouse model has a knock-in mutation in the FKRP gene and develops skeletal, respiratory, and cardiac muscle disease. METHODS: We studied the natural history of the P448Lneo- mouse model over 9 months and the effects of twice weekly treadmill running. Forelimb and hindlimb grip strength (Columbus Instruments) and overall activity (Omnitech Electronics) assessed skeletal muscle function. Echocardiography was performed using VisualSonics Vevo 770 (FujiFilm VisualSonics). Plethysmography was performed using whole body system (ADInstruments). Histological evaluations included quantification of inflammation, fibrosis, central nucleation, and fiber size variation. RESULTS: P448Lneo- mice had significantly increased normalized tissue weights compared to controls at 9 months of age for the heart, gastrocnemius, soleus, tibialis anterior, quadriceps, and triceps. There were no significant differences seen in forelimb or hindlimb grip strength or activity monitoring in P448Lneo- mice with or without exercise compared to controls. Skeletal muscles demonstrated increased inflammation, fibrosis, central nucleation, and variation in fiber size compared to controls (p \u3c 0.05) and worsened with exercise. Plethysmography showed significant differences in respiratory rates and decreased tidal and minute volumes in P448Lneo- mice (p \u3c 0.01). There was increased fibrosis in the diaphragm compared to controls (p \u3c 0.01). Echocardiography demonstrated decreased systolic function in 9-month-old mutant mice (p \u3c 0.01). There was increased myocardial wall thickness and mass (p \u3c 0.001) with increased fibrosis in 9-month-old P448Lneo- mice compared to controls (p \u3c 0.05). mRNA expression for natriuretic peptide type A (Nppa) was significantly increased in P448Lneo- mice compared to controls at 6 months (p \u3c 0.05) and for natriuretic peptide type B (Nppb) at 6 and 9 months of age (p \u3c 0.05). CONCLUSIONS: FKRP-deficient P448Lneo- mice demonstrate significant deficits in cardiac and respiratory functions compared to control mice, and this is associated with increased inflammation and fibrosis. This study provides new functional outcome measures for preclinical trials of FKRP-related muscular dystrophies

The proton pump inhibitor lansoprazole improves the skeletal phenotype in dystrophin deficient mdx mice

Background In Duchenne muscular dystrophy (DMD), loss of the membrane stabilizing protein dystrophin results in myofiber damage. Microinjury to dystrophic myofibers also causes secondary imbalances in sarcolemmic ion permeability and resting membrane potential, which modifies excitation-contraction coupling and increases proinflammatory/apoptotic signaling cascades. Although glucocorticoids remain the standard of care for the treatment of DMD, there is a need to investigate the efficacy of other pharmacological agents targeting the involvement of imbalances in ion flux on dystrophic pathology. Methodology/Principal Findings We designed a preclinical trial to investigate the effects of lansoprazole (LANZO) administration, a proton pump inhibitor, on the dystrophic muscle phenotype in dystrophin deficient (mdx) mice. Eight to ten week-old female mice were assigned to one of four treatment groups (n = 12 per group): (1) vehicle control; (2) 5 mg/kg/day LANZO; (3) 5 mg/kg/day prednisolone; and (4) combined treatment of 5 mg/kg/day prednisolone (PRED) and 5 mg/kg/day LANZO. Treatment was administered orally 5 d/wk for 3 months. At the end of the study, behavioral (Digiscan) and functional outcomes (grip strength and Rotarod) were assessed prior to sacrifice. After sacrifice, body, tissue and organ masses, muscle histology,in vitro muscle force, and creatine kinase levels were measured. Mice in the combined treatment groups displayed significant reductions in the number of degenerating muscle fibers and number of inflammatory foci per muscle field relative to vehicle control. Additionally, mice in the combined treatment group displayed less of a decline in normalized forelimb and hindlimb grip strength and declines in in vitro EDL force after repeated eccentric contractions. Conclusions/Significance Together our findings suggest that combined treatment of LANZO and prednisolone attenuates some components of dystrophic pathology in mdx mice. Our findings warrant future investigation of the clinical efficacy of LANZO and prednisolone combined treatment regimens in dystrophic pathology

Discordance Interpretation of Left Ventricular Size Between Echocardiography and Cardiac Magnetic Resonance in Pediatric Patients With Aortic/Mitral Regurgitation

PURPOSE: This study investigated discordance between echocardiography (echo) and cardiac magnetic resonance (CMR) measurements of the left ventricle (LV) in pediatric patients with aortic and/or mitral regurgitation (AR/MR). METHODS: Retrospective cohort study of pediatric patients. The cohorts were comprised of patients with AR/MR vs. non-AR/MR. Left ventricular end diastolic volume (LVEDV) by CMR and left ventricular internal diameter diastolic (LVIDd) by echo were obtained from clinical reports then echo images were reviewed to remeasure LVEDV by bullet method. Left ventricular internal diameter systolic (LVIDs) and left ventricular ejection fraction (LVEF) measurements by echo and LVEF by CMR were obtained from clinical reports. Fractional shortening (FS%) was recalculated. Z-scores were calculated using normative data. Correlation between echo and CMR LV measurements was assessed using correlation coefficients. Bland-Altman plots assessed bias between imaging modalities. Receiver operator characteristic (ROC) analysis was performed for detection of LV enlargement and LV dysfunction. RESULTS: AR/MR patients had greater discrepancy in LV size interpretation by Z-score compared to non-AR/MR patients. This discrepancy persisted when the bullet method short axis measurements were incorporated. There was negative bias in echo-based measurements compared to CMR. The diagnostic performance of echo in identifying moderate LV enlargement was worse for AR/MR pediatrics patients. CONCLUSION: The discordant interpretation of LV size by echo compared to CMR is worse in pediatric patients with AR/MR when compared to patients without AR/MR even when short axis measurements are incorporated. This finding suggests non-uniform geometrical changes in the LV as it enlarges due to AR/MR